Wiesneriomyces a New Lineage of Dothideomycetes (Ascomycota) Basal to Tubeufiales
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Phytotaxa 176 (1): 283–297 ISSN 1179-3155 (print edition) www.mapress.com/phytotaxa/ Article PHYTOTAXA Copyright © 2014 Magnolia Press ISSN 1179-3163 (online edition) http://dx.doi.org/10.11646/phytotaxa.176.1.27 Wiesneriomyces a new lineage of Dothideomycetes (Ascomycota) basal to Tubeufiales SATINEE SUETRONG¹, NATTAWUT RUNGJINDAMAI¹, SUJINDA SOMMAI2, PATHITA RUNG- AREERATE3, SAYANH SOMMRITHIPOL2 & E.B. GARETH JONES4* 1 Fungal Biodiversity Laboratory (BFBD), National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phahonyothin Road, KhlongNueng, KhlongLuang, PathumThani 12120, Thailand 2 Microbe Interaction Laboratory (BMIT), National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phahonyothin Road, KhlongNueng, KhlongLuang, PathumThani, Thailand 3 Genome Institute, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phahonyothin Road, Klong 1, KlongLuang, 12120, Thailand 4 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia * Email: [email protected] Abstract Wiesneriomyces is an asexual genus comprising two species, found growing on submerged leaves and terrestrial leaf litter in tropical habitats. The genus is characterized by conidiomata with setae, macronematous and branched conidiophores, and hyaline conidia in uniseriate chains connected by narrow isthmi. A multigene (SSU & LSU) analysis of 13 strains of Wiesneriomyces including W. conjuntosporus and W. laurinus formed a monophyletic clade in the Dothideomycetes with high support (98 BSMP, 99 BSML & 1.00 BYPP). The Wiesneriomyces clade is elevated to a higher taxonomic rank, the family Wiesneriomycetaceae, based on cultural, morphological and multi-gene phylogenetic evidence. The family forms a sister lineage to Tubeufiales with strong support. Key words: hyphomycetes, LSU rDNA, new lineage, SSU rDNA, Wiesneriomycetaceae Introduction Some 60% of hyphomycetes (1,727 species) have no known sexual stages (Hyde et al. 2011), although recently molecular techniques have linked a number asexual taxa to families, orders or classes in the ascomycetes and basidiomycetes (Abdel-Wahab et al. 2010, Shenoy et al. 2007, Dai et al. 2012, Diederich et al. 2012, Rungjindamai et al. 2012, Zhang et al. 2012, Hyde et al. 2013). However, fewer than 90% of freshwater hyphomycetes have been linked to sexual morphs. In an ongoing investigation of tropical hyphomycetes and coelomycetes (Sivichai & Jones 2003, Plaingam et al. 2003, Pinruan et al. 2004, 2008, Somrithipol & Jones 2006, Somrithipol et al. 2006, 2007, 2008, Pinnoi et al. 2007, Rungjindamai et al. 2008, Jones et al. 2008), we have employed molecular data to determine their phylogenetic relationships. In this study we report on the genus Wiesneriomyces, which grows on leaf litter in temperate and tropical forests. The genus Wiesneriomyces was introduced by Koorders (1907) with W. javanicus Koord., as the type species. However, this fungus had previously been described 40 years earlier under the name Volutellaria laurina Tassi (Tassi 1897). Clements & Shear (1931) proposed Chaetosira Clem. for W. javanica Koord., but Bisby (1949) pointed out that Chaetosira was an invalid change. Volutellaria laurina Tassi was described from Laurus nobilis leaf litter by Tassi (1897). Soon after describing the fungus, Saccardo (in Tassi 1898) introduced the genus Chaetopeltis Sacc. and transferred V. laurina to the genus as Chaetopeltis laurina (Tassi) Sacc. Sydow & Sydow (1919) found that the fungal genus Chaetopeltis is the later homonym of the algal genus Chaetopeltis Berth. Therefore, they proposed a new genus Tassia H. & P. Syd. and made a new combination, Tassia laurina (Tassi) H. & P. Syd. Accepted by Kevin Hyde: 13 Mar. 2014; published: 20 Aug. 2014 283 Licensed under a Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0 Kirk (1984) examined the holotype of V. laurina and opinioned that it should not be placed in the genus Volutellaria because its conidial morphology clearly differs from Volutellaria acaroids, the type species. Based on priority the generic name Wiesneriomyces is valid. Kirk (1984) proposed a new combination: Wiesneriomyces laurinus (Tassi) P.M. Kirk with V. laurina, Wiesneriomyces javanicus, Chaetopeltis laurina (Tassi) Sacc. and Tassia laurina (Tassi) H. & P. Syd. as synonyms. Subsequently, Kuthubutheen & Nawawi (1988) described W. conjunctosporus on submerged leaf litter from Pasoh Forest Reserve, Malaysia. Both species have been recently collected in Thailand and isolated into axenic culture. These form the basis for this phylogenetic study of the genus Wiesneriomyces. Wiesneriomyces is characterized by conidiomata with thick-walled setae, macronematous and branched conidiophores, and hyaline conidia in uniseriate chains connected by narrow isthmi. Based on the original discussion, W. conjunctosporus differs from W. laurinus as it has larger setae, conidiomata and conidia and longer conidial chains (Kuthubutheen & Nawawi, 1988). However, a number of specimens collected later reveal the measurements of the two species overlapped. The most distinctive morphology to separate these species is the structure of the conidiomata. The conidioma of Wiesneriomyces laurinus is a prominent sporodochium with conidiophores and setae arising from its basal, pseudoparenchymatous stalk. On the other hand, the conidioma of W. conjunctosporous is a less prominent sporodochium encircled by a single row of setae which arise from the immersed mycelium. Genera producing setose, pigmented sporodochia, conidiophores, and isthmospores resembling Wiesneriomyces include Abgliophragma R.Y. Roy & S. Gujarati and Gliophragma Subram. & B.C. Lodha. Gliophragma differs from Wiesneriomyces in possessing larger setae attached to the synnemata. Pirozynski (1972) and Matsushima (1971) treated Gliophragma as a synonym of Wiesneriomyces. Phalangispora Nawawi & Webster (1982) differs from Wiesneriomyces in having branched chains of conidia. All Wiesneriomyces species have been collected from terrestrial leaf litter. Wiesneriomyces conjunctosporus was also collected from submerged litter in a stream (Kuthubutheen & Nawawi, 1988). Wiesneriomyces laurinus is widely reported from Java (Koorders 1907), India (Subramanian 1956), Panama (Manotis & Strain 1968), Papua New Guinea (Matsushima 1971), UK (Kirk 1983), Taiwan (Matsushima 1980, Australia (Shaw & Sutton 1985) and Malaysia (Kuthubutheen & Nawai 1988), whereas W. conjunctosporus is know only from Malaysia and Thailand. Materials and methods Fungal cultures and maintenance Two species of Wiesneriomyces were collected from Thailand. This comprised seven strains of strains of W. conjunctosporus and six strains of W. laurinus. These Wiesneriomyces species were isolated by single spore methodology as outlined by Chomnunti et al. (2011) from various locations and on separate occasions in Thailand (Table 1). All cultures are deposited at BIOTEC Culture Collection (BCC), their BCC numbers and accession numbers of two rDNA sequences are shown in Table 1. DNA Extraction and PCR amplification The cultures were grown on potato dextrose agar (PDA) and incubated at room temperature for two weeks. Actively growing mycelia were harvested en masse and placed in a 1.5 ml Eppendorf tube. Genomic DNA was extracted using a CTAB method (O'Donnell et al. 1997) which was modified and previously described by Rungjindamai et al. (2012). The purified genomic DNA was used as a DNA template for PCR amplification. Two regions of rDNA sequences including the small subunit (SSU) and large subunit (LSU) were amplified using primers for NS1, NS3, NS4 and NS6 (for SSU) and JS1 and LR7 (for LSU) (White et al. 1990, Bunyard et al. 1994) using DyNAzyme II DNA polymerase kit (Fizzymes, Espoo, Finland). PCR amplification was performed using a DNA Engine DYAD ALD 1244 Thermocycler (MJ Research, the US). The PCR conditions were 94°C for 3 min, followed by 35 cycles of 94°C for 1 min, 49°C for 1 min and 72°C for 1 min 30 sec, a final extension at 72°C for 8 min and held at 25°C. The PCR products were purified with NucleoSpin Extract DNA purification kit (Macherey- Nagel, Germany) and sequenced by Macrogen Inc. (South Korea) using the same primers as for amplification. Sequence alignment and phylogenetic analysis SSU and LSU DNA sequences of 13 Wiesneriomyces strains were compared to sequences deposited in GenBank using the BLAST search tool to obtain the closest matched sequences (Altschul et al. 1990). Additional 284 • Phytotaxa 176 (1) © 2014 Magnolia Press SUETRONG ET AL. representative taxa from the Dothideomycetes appearing in previously published papers were added into the dataset (Suetrong et al. 2011, Jones et al. 2012, Hyde et al. 2013). The SSU and LSU sequences were multiple aligned using Clustal W 1.6 (Thompson et al. 1994) and adjusted manually where necessary using BioEdit 7.5.0.3 (Hall 2006). Manual gap adjustments were made to improve the alignment. Ambiguously aligned regions were excluded. Missing data at the 5′-and 3′-end of partial sequences were coded by a ‘?’. The final alignment was again optimized by eye and manually corrected using Se-Al v. 2.0a8 (Rambaut 1996). The tree construction procedure was performed in PAUP* 4.0b10 (Swofford 2002). Phylogenetic trees were visualized using the program Treeview (Page 1996). The phylogenetic analyses of different datasets were performed using maximum parsimony, Bayesian